The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.